Mammalian Caspases Research Articles

Mycoplasma bovis activates apoptotic caspases to suppress xenophagy for its intracellular survival

Mammalian caspases are categorized into apoptotic and inflammatory types. Apoptotic caspases mediate apoptosis activation, while inflammatory caspases participate in inflammasome activation. Previous studies have shown that apoptotic caspases regulate autophagy in both cancer and pharmacological treatment models. However, the relationship between apoptotic caspases and xenophagy during pathogen infection remains elusive. In the current study, we used Mycoplasma bovis (M. bovis) as a model pathogen investigating the relationship between apoptotic caspases and xenophagy during infection. We found that M. bovis activated apoptotic caspases by triggering mitochondrial damage in macrophages, and the intracellular survival of M. bovis was enhanced by the activation of apoptotic caspases and restricted by the inhibition of apoptotic caspases. Moreover, confocal microscopy and Western blot analysis revealed that the activation of apoptotic caspases impedes host xenophagy by cleaving autophagy-related protein Beclin 1. Our findings indicate that M. bovis utilizes host apoptotic caspases to suppress xenophagy, thereby enhancing its intracellular survival. This research contributes to understanding the interplay between apoptotic caspases and xenophagy during pathogen infection, offering novel insights into the intracellular survival mechanisms of mycoplasma in macrophages.

The prodomain of Arabidopsis metacaspase 2 positively regulates immune signaling mediated by pattern-recognition receptors.

Metacaspases (MCs) are structural homologs of mammalian caspases found in plants, fungi, and protozoa. Type-I MCs carry an N-terminal prodomain, the function of which is unclear. Through genetic analysis of Arabidopsis mc2-1, a T-DNA insertion mutant of MC2, we demonstrated that the prodomain of metacaspase 2 (MC2) promotes immune signaling mediated by pattern-recognition receptors (PRRs). In mc2-1, immune responses are constitutively activated. The receptor-like kinases (RLKs) BAK1/BKK1 and SOBIR1 are required for the autoimmune phenotype of mc2-1, suggesting that immune signaling mediated by the receptor-like protein (RLP)-type PRRs is activated in mc2-1. A suppressor screen identified multiple mutations in the first exon of MC2, which suppress the autoimmunity in mc2-1. Further analysis revealed that the T-DNA insertion at the end of exon 1 of MC2 causes elevated expression of the MC2 prodomain, and overexpression of the MC2 prodomain in wild-type (WT) plants results in the activation of immune responses. The MC2 prodomain interacts with BIR1, which inhibits RLP-mediated immune signaling by interacting with BAK1, suggesting that the MC2 prodomain promotes plant defense responses by interfering with the function of BIR1. Our study uncovers an unexpected function of the prodomain of a MC in plant immunity.

CaBir1 functions as an inhibitor-of-apoptosis and affects caspase-like activitiy in Candida albicans

CaMca1 is the only metacaspase in Candida albicans, which shows structural homology to the mammalian caspases. CaMca1 consists of the caspase domain, the P20 and P10 regions, and the conserved catalytic histidine-cysteine dyad that is required for executing apoptosis in C. albicans. However, little is known about the proteolytic processing of CaMca1 or its activation under apoptosis-inducing conditions. To understand the regulation of this process, we characterized CaBir1 which is the single IAP (inhibitor-of-apoptosis protein) in C. albicans. IAPs are a family of proteins whose members all harbor a BIR (baculovirus IAP repeat) domain and negatively regulate apoptosis by inhibiting caspases. We found that the Cabir1/Cabir1 deletion mutant exhibited increased apoptotic phenotypes, such as ROS accumulation, nuclear segmentation, and cell survival, under apoptosis-inducing conditions. Examination of CaMca1 cleavage patterns in response to various apoptotic stresses revealed that these cleavages were stress-specific and dependent on the catalytic histidine-cysteine residues of CaMca1. The Cabir1/Cabir1 mutation was not associated with altered CaMca1 processing with or without apoptotic stimuli, but the Cabir1/Cabir1 mutant exhibited significantly increased caspase-like activities. These results suggest that CaBir1 acts as an apoptosis inhibitor by regulating caspase-like activity, but not CaMca1 processing.

The Role of Caspase-2 in Regulating Cell Fate.

Caspase-2 is the most evolutionarily conserved member of the mammalian caspase family and has been implicated in both apoptotic and non-apoptotic signaling pathways, including tumor suppression, cell cycle regulation, and DNA repair. A myriad of signaling molecules is associated with the tight regulation of caspase-2 to mediate multiple cellular processes far beyond apoptotic cell death. This review provides a comprehensive overview of the literature pertaining to possible sophisticated molecular mechanisms underlying the multifaceted process of caspase-2 activation and to highlight its interplay between factors that promote or suppress apoptosis in a complicated regulatory network that determines the fate of a cell from its birth and throughout its life.

Antifilarial efficacy of green silver nanoparticles synthesized using Andrographis paniculata

The currently available antifilarial drugs are mostly effective against microfilariae. Antifilarial drugs have disadvantages such as toxicity and the development of resistance due to the continuous use. Therefore alternative drugs are required for the control of disease. At present, nanoparticles are used for developing anti-parasitic therapy for their special properties such as smallest in size, bio-availability, bio-compatibility and penetration capacity into a cell. In the present study green nanoparticles were biosynthesized by using leaf extract of Andrographis paniculata to evaluate its antifilarial efficacy. Biosynthesized nanoparticles were characterized by UV–Vis spectroscopy, FTIR, XRD, SEM, EDAX, TEM, HRTEM, AFM and found to be homogenous with average size of 11 nm. The antifilarial efficacy of green nanoparticles was based on adult filarial parasite motility and viability assay. These green nanoparticles (NPs) were found to have antifilarial activity with LC50 of 11.6 μg/ml against adult female filarial parasites. The green nanoparticles induced oxidative stress as evidenced by elevated ROS production and decline of parasitic GST, GR, TRxR and GSH levels in the parasite. The activation of ced-3 gene, a homolog of mammalian caspase 3, reduced expression of ced-9 and decreased activity of cytochrome c oxidase suggested induction of mitochondrial mediated apoptosis in parasite. These green NPs are more effective than the plant extract. This is the first report where green nanoparticles synthesized from A. paniculata showed antifilarial efficacy against adult filarial parasites.

Combination Suicide Gene Delivery with an Adeno-Associated Virus Vector Encoding Inducible Caspase-9 and a Chemical Inducer of Dimerization Is Effective in a Xenotransplantation Model of Hepatocellular Carcinoma.

Current treatment approaches for hepatocellular carcinoma (HCC) have a narrow therapeutic index and alternate modes of treatment are thus required. We have utilized a gene delivery vector containing inducible caspase 9 (iCasp9) gene, which is a synthetic analogue based on the mammalian caspase 9 and fused to a human FK506 binding protein that allows its conditional dimerization to a synthetic, small molecule [chemical inducer of dimerization, AP20187] and results in target cell apoptosis. In our studies, we have tested these synthetic vectors based on an adeno-associated virus platform for their potential anti-tumorigenic effect in human HCC cells in vitro and in a HCC tumor model developed in nude mice. Our data demonstrates that the iCasp9-AP20187 bioconjugate is able to trigger terminal effectors of cellular apoptosis and presents a viable approach for the potential treatment of HCC.

Inflammatory caspase regulation: maintaining balance between inflammation and cell death in health and disease.

Members of the mammalian inflammatory caspase family, including caspase-1, caspase-4, caspase-5, caspase-11, and caspase-12, are key regulators of the innate immune response. Most studies to date have focused on the role of caspase-1 in the maturation of the proinflammatory cytokine interleukin-1β and its upstream regulation by the inflammasome signaling complexes. However, an emerging body of research has supported a role for caspase-4, caspase-5, and caspase-11 in both regulating caspase-1 activation and inducing the inflammatory form of cell death called pyroptosis. This inflammatory caspase pathway appears essential for the regulation of cytokine processing. Consequently, insight into this noncanonical pathway may reveal important and, to date, understudied targets for the treatment of autoinflammatory disorders where the inflammasome pathway is dysregulated. Here, we will discuss the mechanisms of inflammasome and inflammatory caspase activation and how these pathways intersect to promote pathogen clearance.

The Caspase-3 homolog DrICE regulates endocytic trafficking during Drosophila tracheal morphogenesis

Although well known for its role in apoptosis, the executioner caspase DrICE has a non-apoptotic function that is required for elongation of the epithelial tubes of the Drosophila tracheal system. Here, we show that DrICE acts downstream of the Hippo Network to regulate endocytic trafficking of at least four cell polarity, cell junction and apical extracellular matrix proteins involved in tracheal tube size control: Crumbs, Uninflatable, Kune-Kune and Serpentine. We further show that tracheal cells are competent to undergo apoptosis, even though developmentally-regulated DrICE function rarely kills tracheal cells. Our results reveal a developmental role for caspases, a pool of DrICE that co-localizes with Clathrin, and a mechanism by which the Hippo Network controls endocytic trafficking. Given reports of in vitro regulation of endocytosis by mammalian caspases during apoptosis, we propose that caspase-mediated regulation of endocytic trafficking is an evolutionarily conserved function of caspases that can be deployed during morphogenesis.

Caenorhabditis elegans ced-3 Caspase Is Required for Asymmetric Divisions That Generate Cells Programmed To Die.

Caspases have functions other than in apoptosis. Here, we report that Caenorhabditis elegans CED-3 caspase regulates asymmetric cell division. Many of the 131 cells that are “programmed” to die during C. elegans development are the smaller daughter of a neuroblast that divides asymmetrically by size and fate. We have previously shown that CED-3 caspase is activated in such neuroblasts, and that before neuroblast division, a gradient of CED-3 caspase activity is formed in a ced-1 MEGF10 (multiple EGF-like domains 10)-dependent manner. This results in the nonrandom segregation of active CED-3 caspase or “apoptotic potential” into the smaller daughter. We now show that CED-3 caspase is necessary for the ability of neuroblasts to divide asymmetrically by size. In addition, we provide evidence that a pig-1 MELK (maternal embryonic leucine zipper kinase)-dependent reciprocal gradient of “mitotic potential” is formed in the QL.p neuroblast, and that CED-3 caspase antagonizes this mitotic potential. Based on these findings, we propose that CED-3 caspase plays a critical role in the asymmetric division by size and fate of neuroblasts, and that this contributes to the reproducibility and robustness with which the smaller daughter cell is produced and adopts the apoptotic fate. Finally, the function of CED-3 caspase in this context is dependent on its activation through the conserved egl-1 BH3-only, ced-9 Bcl-2, and ced-4 Apaf-1 pathway. In mammals, caspases affect various aspects of stem cell lineages. We speculate that the new nonapoptotic function of C. elegans CED-3 caspase in asymmetric neuroblast division is relevant to the function(s) of mammalian caspases in stem cells.

Starfish Apaf-1 activates effector caspase-3/9 upon apoptosis of aged eggs

Caspase-3-related DEVDase activity is initiated upon apoptosis in unfertilized starfish eggs. In this study, we cloned a starfish procaspase-3 corresponding to mammalian effector caspase containing a CARD that is similar to the amino terminal CARD of mammalian capsase-9, and we named it procaspase-3/9. Recombinant procaspase-3/9 expressed at 15 °C was cleaved to form active caspase-3/9 which has DEVDase activity. Microinjection of the active caspase-3/9 into starfish oocytes/eggs induced apoptosis. An antibody against the recombinant protein recognized endogenous procaspase-3/9 in starfish oocytes, which was cleaved upon apoptosis in aged unfertilized eggs. These results indicate that caspase-3/9 is an effector caspase in starfish. To verify the mechanism of caspase-3/9 activation, we cloned starfish Apaf-1 containing a CARD, a NOD, and 11 WD40 repeat regions, and we named it sfApaf-1. Recombinant sfApaf-1 CARD interacts with recombinant caspase-3/9 CARD and with endogenous procaspase-3/9 in cell-free preparations made from starfish oocytes, causing the formation of active caspase-3/9. When the cell-free preparation without mitochondria was incubated with inactive recombinant procaspase-3/9 expressed at 37 °C, DEVDase activity increased and apoptosome-like complexes were formed in the high molecular weight fractions containing both sfApaf-1 and cleaved caspase-3/9. These results suggest that sfApaf-1 activation is not dependent on cytochrome c.

Characterization of the XIAP-Inhibiting Proanthocyanidin Fraction of the Aerial Parts of Ephedra sinica.

The X-linked inhibitor of apoptosis protein is a cellular protein that inhibits the activity of mammalian caspases and promotes resistance to apoptosis. The ethanol extract of the aerial parts of Ephedra sinica has been identified to possess inhibitory activity of the X-linked inhibitor of apoptosis protein by an in vitro fluorescence polarization assay using the BIR3 domain of the X-linked inhibitor of apoptosis protein. Bioactivity-guided fractionation identified proanthocyanidin-enriched fractions as the active principles. The most active fraction showed an IC50 value of 27.3 µg/mL (CI95: 25.9-28.9 µg/mL) corresponding to 9.6 µM (CI95: 9.1-10.1 µM) calculated by the use of the determined average molecular weight of 2853.5. Samples were analyzed by a thiolytic degradation/HPLC-MS assay, UHPLC-HRMS, and 1D NMR.The thiolytic degradation/HPLC-MS assay revealed a mean degree of polymerization of 9.5 ± 0.2 units (calculated average MW 2853.5) for the active fraction and 11.4 ± 0.6 units (calculated average MW 3437.0) for the most related inactive fraction. Chemical characterization identified (epi)gallocatechin (76.6 ± 1.0 % active; 80.7 ± 2.7 % inactive sample) and (epi)catechin units as building blocks. Interestingly, the investigated proanthocyanidins turned out to be a complex mixture of double linked A-type (binding 2-O-7″, 4-6″) and single linked B-type units.This study identified oligomeric proanthocyanidins as active principles of E. sinica in vitro by a fluorescence polarization assay and via protein fragment complementation analysis.

Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome

Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys147, resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.

Role and Association of Inflammatory and Apoptotic Caspases in Renal Tubulointerstitial Fibrosis

Background/Aims: Caspases, an evolutionary conserved family of aspartate-specific cystein proteases, play pivotal roles in apoptotic and inflammatory signaling. Thus far, 14 mammalian caspases are identified and categorized into 3 distinct sub-types: inflammatory caspases, apoptotic initiator and apoptotic executioner. Caspase-1 is an inflammatory caspase, while caspase-7 belongs to apoptotic executioner. The roles and association of these two distinct types of caspases in renal tubulointerstitial fibrosis (TIF) have not been well recognized. Methods: Caspase-1 inhibitor Z-YVAD-FMK and caspase-7 siRNA were used in tubular epithelial cell line NRK-52E (TECs) to test their effects on transforming growth factor-beta1 (TGF-β1) stimulation. In vivo, Unilateral ureteral obstruction (UUO) animal model was employed in wild-type (WT) and caspase-1 knock out (KO) (caspase-1^-/-) mice. Results: In current study, we found that caspase-7 was obviously activated in cultured TECs stimulated by TGF-β1 and in UUO model of WT mice. While in UUO model of caspase-1 KO mice, the increased caspase-7 activation was suppressed significantly along with reduced trans-differentiation and minimized extracellular matrix (ECM) accumulation, as demonstrated by western blot, Masson trichrome staining and immunohistochemistry. In addition, pharmacological inhibition of caspase-1 dampened caspase-7 activation and TECs' transdifferentiation induced by TGF-β1 exposure, which was consistent with in vivo study. Notably, caspase-7 gene knock down by specific siRNA abrogated TGF-β1 driven TECs' trans-differentiation and reduced ECM accumulation. Conclusions: Our study associated inflammatory and apoptotic caspases in TIF for the first time and we further confirmed that caspase-1 activation is an upstream event of apoptotic caspase-7 induction in TIF triggered by UUO and in TECs mediated by TGF-β1 induced transdifferentiation.

Strategies for tracking anastasis, a cell survival phenomenon that reverses apoptosis.

Anastasis (Greek for "rising to life") refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensorfor identifying and tracking anastasis in mammalian cells.

An in silico appraisal to identify high affinity anti-apoptotic synthetic tetrapeptide inhibitors targeting the mammalian caspase 3 enzyme.

Apoptosis is a general phenomenon of all multicellular organisms and caspases form a group of important proteins central to suicide of cells. Pathologies like cancer, Myocardial infarction, Stroke, Sepsis, Alzheimer's, Psoriasis, Parkinson and Huntington diseases are often associated with change in caspase 3 mediated apoptosis and therefore, caspases may serve as potential inhibitory targets for drug development. In the present study, two series of synthetic acetylated tetrapeptides containing aldehyde and fluromethyl keto groups respectively at the C terminus were proposed. All these compounds were evaluated for binding affinity against caspase 3 structure. In series 1 compound Ac-DEHD-CHO demonstrated appreciable and high binding affinity (Rerank Score: -138.899) against caspase 3. While in series 2 it was Ac-WEVD-FMK which showed higher binding affinity (Rerank Score: -139.317). Further these two compounds met ADMET properties and demonstrated to be non- toxic.

Old, new and emerging functions of caspases.

Caspases are proteases with a well-defined role in apoptosis. However, increasing evidence indicates multiple functions of caspases outside apoptosis. Caspase-1 and caspase-11 have roles in inflammation and mediating inflammatory cell death by pyroptosis. Similarly, caspase-8 has dual role in cell death, mediating both receptor-mediated apoptosis and in its absence, necroptosis. Caspase-8 also functions in maintenance and homeostasis of the adult T-cell population. Caspase-3 has important roles in tissue differentiation, regeneration and neural development in ways that are distinct and do not involve any apoptotic activity. Several other caspases have demonstrated anti-tumor roles. Notable among them are caspase-2, -8 and -14. However, increased caspase-2 and -8 expression in certain types of tumor has also been linked to promoting tumorigenesis. Increased levels of caspase-3 in tumor cells causes apoptosis and secretion of paracrine factors that promotes compensatory proliferation in surrounding normal tissues, tumor cell repopulation and presents a barrier for effective therapeutic strategies. Besides this caspase-2 has emerged as a unique caspase with potential roles in maintaining genomic stability, metabolism, autophagy and aging. The present review focuses on some of these less studied and emerging functions of mammalian caspases.

Effect of CDNB on filarial thioredoxin reductase : A proteomic and biochemical approach.

Lymphatic filariasis is one of the most prevalent tropical diseases caused by tissue dwelling parasitic nematodes viz., Wuchereria bancrofti, Brugia malayi and Brugia timori. Currently available antifilarial drugs effectively eliminate larval stages of the parasite but are ineffective against the adult worms. Therefore, there is an urgent need for finding proteins/enzymes which play a crucial role in the persistence of these parasites. Our study for the first time reports the important role played by S. cervi TrxR in its survival. Thus, suggesting filarial TrxR as a potent chemotherapeutic target against lymphatic filariasis. This would help in screening of new compounds having macrofilaricidal activity.

Involvement of zebrafish RIG-I in NF-κB and IFN signaling pathways: Insights into functional conservation of RIG-I in antiviral innate immunity

The retinoic acid-inducible gene I (RIG-I) is a critical sensor for host recognition of RNA virus infection and initiation of antiviral signaling pathways in mammals. However, data on the occurrence and functions of this molecule in lower vertebrates are limited. In this study, we characterized an RIG-I homolog (DrRIG-I) from zebrafish. Structurally, this DrRIG-I shares a number of conserved functional domains/motifs with its mammalian counterparts, namely, caspase activation and recruitment domain, DExD/H box, a helicase domain, and a C-terminal domain. Functionally, stimulation with DrRIG-I CARD in zebrafish embryos significantly activated the NF-κB and IFN signaling pathways, leading to the expression of TNF-α, IL-8 and IFN-induced Mx, ISG15, and viperin. However, knockdown of TRIM25 (a pivotal activator for RIG-I receptors) significantly suppressed the induced activation of IFN signaling. Results suggested the functional conservation of RIG-I receptors in the NF-κB and IFN signaling pathways between teleosts and mammals, providing a perspective into the evolutionary history of RIG-I-mediated antiviral innate immunity.

Inhibition of Cathepsin B by E-64 Induces Oxidative Stress and Apoptosis in Filarial Parasite

BackgroundCurrent available antifilarial drug strategies only eliminate the larval stages of filarial parasites. Therefore, there is an urgent need of drugs which are macrofilaricidals. Identification of molecular targets crucial for survival of parasite is a prerequisite for drug designing. Cathepsin B, a cysteine protease family member is known to play crucial role in the normal growth, digestion of nutrients, exsheathment of the helminth parasites. Therefore, we targeted this enzyme in the filarial parasite using its specific inhibitor, E-64.Methods and FindingsWe have exposed the parasites to E-64 and observed their motility and viability at various time intervals. It caused marked decrease in the motility and viability of the parasites ultimately leading to their death after 8 hours. It is well known that E-64 protects the cell from apoptosis, however, it causes apoptotic effect in carcinoma cell lines. To understand the mechanism of action of E-64 on parasite survival, we have measured levels of different apoptotic markers in the treated parasites. E-64 significantly reduced the level of ced-9 and activity of tyrosine phosphatases, cytochrome c oxidase. It also activated ced-3, homolog of mammalian caspase 3 suggesting initiation of an apoptotic like event in the filarial parasites. Different antioxidant enzymes were also evaluated to further explore the mechanism behind the death of the parasites. There was marked decrease in the level of GSH and activity of Glutathione reductase and glutathione-s-transferase leading to increased generation of reactive oxygen species. This led to the induced oxidation of fatty acids and protein which might alter the mitochondrial membrane permeability.ConclusionThis study suggests that inhibition of cathepsin B by E-64 generates oxidative stress followed by mitochondrial mediated apoptotic like event in filarial parasites leading to their death. Hence, suggesting filarial cathepsin B as a potential chemotherapeutic target for lymphatic filariasis.

砷诱导蚕豆气孔保卫细胞死亡效应研究

PDF HTML阅读 XML下载 导出引用 引用提醒 砷诱导蚕豆气孔保卫细胞死亡的毒性效应 DOI: 10.5846/stxb201210171445 作者: 作者单位: 山西大学生命科学学院,山西大学 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金项目（30870454，30470318）；教育部高等学校博士学科点基金项目（20070108007，20121401110007）；山西省回国留学人员科研资助项目（2012013） Arsenic induces guard cell death in leaf epidermis of Vicia faba Author: Affiliation: School of Life Science,Shanxi University,Shanxi University Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:采用蚕豆（Vicia faba L.）叶面气孔保卫细胞，研究砷对细胞的毒性效应。结果表明，0.3-10 mg/L的NaAsO2能降低保卫细胞活性，使部分细胞死亡，死亡率随砷浓度升高而增高。死细胞中呈现核固缩、核崩解等典型程序性死亡特征，且泛caspase抑制剂Z-Asp-CH2-DCB能阻止NaAsO2诱发的细胞死亡。过氧化氢清除剂过氧化氢酶与NaAsO2共同作用时，细胞死亡率显著低于砷单独处理组，保卫细胞内Ca2+水平降低，具程序性死亡特征的细胞数减少；Ca2+特异性螯合剂EGTA亦能降低NaAsO2诱发的细胞死亡。研究结果表明，NaAsO2能诱发蚕豆保卫细胞程序性死亡，该过程由胁迫引发的ROS升高引起，ROS可能通过激活质膜Ca2+通道，使胞外Ca2+内流，造成胞内Ca2+浓度升高，进而诱导细胞程序性死亡。 Abstract:Arsenic is a highly toxic metalloid for all forms of life including plants. Arsenic enters in plants through phosphate transporters as a phosphate analogue or through aquaglycoporins. Uptake of arsenic in plant tissues can affect plant metabolism, causing various physiological disorders, structural abnormalities and even plant death. Oxidative stress is considered to be a key mechanism of arsenic toxicity.In this study, the effect of sodium arsenite (NaAsO2) on guard cell viability was investigated in detached epidermis of V. faba leaves. Epidermal strips were obtained from 4-week-old plants by peeling off the lower epidermis of V. faba leaves and incubated in 2-(N-morpholino) ethanesulfonic acid (MES) buffer containing some chemicals (NaAsO2 with or without some antagonists) for 3 h in white light at 23 ℃ as the treatments. After treatment, the epidermal strips were stained with fluorescein diacetate (FDA) to show cell viability, or with 2',7'-dichlorofluorescein diacetate (DCFH-DA) and fluo-3 acetomethoxyester (Fluo-3AM) respectively to indicate intracellular reactive oxygen species (ROS) and calcium ion (Ca2+) levels.The results of our experiments showed that NaAsO2 treatment significantly decreased cell viability and induced cell death in the concentration range of 0.3 to 10 mg/L. Arsenic provokes synchronous increases in cell death rate and intracellular levels of ROS and Ca2+ in V. faba guard cells. The typical nuclear morphological changes including nuclear fragmentation and nuclear condensation were observed in As-treated guard cells, while Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB), a specific inhibitor of mammalian caspases, significantly blocked As-induced cell death. The occurrence of characteristic features of programmed cell death (PCD) and the inhibitory effect of caspase inhibitor Z-Asp-CH2-DCB on As-induced cell death suggest the activation of a PCD pathway evoked by arsenic exposure. Application of antioxidant catalase significantly inhibited As-induced cell death, PCD-to-total-cells ratio and intracellular Ca2+ increase provoked by arsenic. A specific Ca2+ chelator ethylene glycol tetraacetic acid also significantly decreased the cell death caused by arsenic. These results clearly demonstrated that arsenic induced cell death associated with obvious increases in intracellular ROS and Ca2+ levels, in which ROS positively regulated intracellular Ca2+ level.ROS generation and intracellular Ca2+ level increase in As-treated stomatal guard cells were involved in the process of As-induced cell death. The results of the present study indicate that arsenite induced guard cell death via a PCD pathway through ROS mediating Ca2+ elevation. 参考文献 相似文献 引证文献